Distal regulatory functions for the uvrC of E. coli

Volume 12 Number 13 1984 Nucleic Acids Research Distal regulatory functions for the uvrC gene of E. coli Surendra Sharma1, Thomas Stark and Robb E.Moses* Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA Received 29 February 1984; Revised and Accepted 8 June 1984 INTRODUCTION The the uvrA, uvrB and uvrC genes control nucleotide excision repair of DNA damage (1,2). Recently, the uvrA, uvrB and uvrC genes have been cloned and characterized (3-9). be complex. The regulation of expression of these genes appears to For example, the uvrA and uvrB genes have been shown to be regulated by the recA-lexA control system, and thus their gene products can be induced in exponentially growing cells via "SOS" response (5,10,11). This is in contrast to the previously held notion that these genes were constitutlvely expressed. Such "SOS"-controlled regulation has not yet been defined for the uvrC gene. We showed that the uvrC gene is located on a 1.9 kb fragment and codes for a protein of 66,000 Mr (7). contain the uvrC gene promoter. The 1.9 kb fragment does not appear to To identify the control regions for the uvrC gene, we subcloned the uvrC structural gene together with 5'-flanklng DNA. We concluded (12) that (a) the 1.9 kb fragment when fused at the 5' end with a heterologous promoter complemented the uvrC defect, (b) an adjacent region of at least 1 kb 5' to the structural gene did not contain an active in vivo uvrC promoter, and (c) a distal regulatory reglon(s) was required for optimal Jjn_ © IRL Press Limited, Oxford, England. 5341 ABSTRACT We find that the uvrC gene is preceded by three promoters (PI, P2 and P3), identified by heparin-reslstant RNA polymerase-DNA complex formation. P2 and P3 promoters are located proximal to the 5' end of the uvrC gene, while the PI promoter is separated from the uvrC structural gene by an interposed DNA region of more than 1 kb. We have reported that P2 and P3 are not sufficient to promote uvrC complementation. However, plasmids containing the direct fusion of the PI promoter to the uvrC gene complements the uvrC defect. Insertion of IS1 downstream from the PI promoter leads to efficient synthesis of the uvrC protein as measured in maxlcells. Fusion of the lac promoter to the uvrC structural gene can substitute for _ln_ vivo regulatory functions. We conclude that uvrC protein synthesis is controlled in a complex manner and that a distal promoter, PI, is required. Nucleic Acids Research vivo complementation of the uvrC defect. Interestingly, a plasmid (pUV7) containing the 1.9 Kb structural region and 2.4 Kb 5' to the gene, failed to promote synthesis of detectable amounts of the uvrC protein under non-induced conditions, although this plasmid confers normal UV resistance to uvrC" cells and codes for a 27,000-Mr protein in addition to uvrC (12). We have carried out fine mapping for the 15. coll RNA polymerase binding sites on the 2.4 kb upstream DNA sequences, and constructed deletion and insertion mutations to identify the presence of putative promoter regions. Our results suggest that the uvrC gene is preceded by three promoters. In addition, we have utilized the lac promoter to bypass the distal promoterWe conclude that a distal regulatory region is involved in in vivo regulation of the uvrC gene. MATERIALS AND METHODS co11 L' strains AB1157 (1), AB1184 uvrC (1), SR57 uvrC , recA~ (6), CSR603 uvrA", recAl, phr~ (13), and AD10 recA~, pgsA (14) , were used as host strains. terial General procedures for construction and cloning of plasmids, bac- transformation, purification analysis were as described (7,12). of plasmid DNA and restriction enzyme Maxi-cell preparations and 35S-methlonine labeling of plasmid encoded proteins were done as reported (15). UV-survival of AB1884 (uvrC~) transformed with plasmids was measured using cell dilutions at different UV-doses (12). Plasmids Plasrald TP88, the source of the lac promoter fragment, was a gift from Dr. Anthony R. Poteete of the University of Massachusetts Medical School. Plastnid8 pUV201 and pUV301 have been previusly described (7,12). Construction of plasmid pUV7-8 required llgation of Hind III linkers. The preparation of "blunt ends" for linker ligatlon was done by filling in protruding ends generated by Bgl II. The digested DNA (5 ug) was incubated with Klenow fragment of DNA polymerase I (15 units) for 15 minutes at 14°C in a reaction mixture (100 ul) containing 60 mM Tris-HCl, pH 7.6, 8 mM MgCl2, 10 mM DTT, 400 uM ATP, and 200 MM dNTPs. by addition of This reaction was immediately followed 32 P-labeled Hind III linkers and by treatment with T4 phage DNA ligase (40 units). Ligatlon was performed by incubating the reaction mixture at 14 °C for 2 hours, and then continuing the Incubation at 4°C overnight. The ligatlon mixture was precipitated and digested with excess Hind III. Required fragments were first identified by autoradiography and isolated for further 5342 mediated regulation effect and to overproduce the uvrC protein. Nucleic Acids Research use. For construction of plasraid pUV7-3, blunt end llgatlon was carried out as described above. RNA Polymerase Binding Assay Reaction conditions for RNA polymerase-DNA interactions were as described (12). In brief, isolated DNA fragments were digested with restriction enzymes to generate smaller fragments. merase (4:1 RNA DNA fragments were Incubated with RNA poly- polymerase :DNA ratio) for 30 minutes at 37 °C Weak RNA polymerase-DNA complexes were further competed out with heparin (200 pg). The proteln-DNA complexes were retained on nitrocellulose filters, eluted and separated on 7.5% acrylamide gel. Transformants containing hybrid plasmlds which contain lac promoters were selected on amplcillin-agar plates containing the indicator 5-chloro-4-bromo3-indolyl-B-D galactoside (XG) (16). The lac operator present on the lac promoter fragment can titrate the cellular repressor allowing synthesis of ggalactosidase (17). The synthesis of f3-galactosldase in transformants gives blue colonies on XG Indicator plates. We purified single colonies on fresh XG plates and selected dark blue colonies. XG solution (20 mg/ml) was prepared in N,N-dimethyl formamide (16), and 50 pi of this solution was spread on agar plates. Materials Growth media were from Difco. Ampicillin, tetracycllne, chloramphenicol, and D-cycloserine, were purchased from Sigma. T4 ligase were obtained Restriction endonucleases and from Bethesda Research Laboratories, New England Biolabs, and Boehringer Biochemicals. RESULTS Multiple Promoters of the uvrC Gene Our previous studies have shown that a plasraid, pUV301, containing the uvrC structural gene and the contiguous 0.9 kb 5' region does not complement the uvrC defect, in contrast to plasmld pUV7 which carries a 1.5 kb additional upstream region with the 0.9 kb fragment (12). This suggested the presence of (...truncated)